Role of ASXL1 mutations in myeloid malignancies Addition of Sex Combs-like 1 (ASXL1) is an Enhancer of Trithorax and Polycomb (ETP) protein as ASXL1 loss in Drosophila causes homeotic transformations and alterations in Trithorax and Polycomb- mediated gene expression. Recently, recurrent mutations in ASXL1 were discovered in patients with myeloid malignancies, including myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and acute myeloid leukemia (AML). Moreover, we have identified from high-throughput sequencing studies of patients with MDS, MPN, and de novo AML that mutations in ASXL1 are associated with significantly poorer overall survival in uniformly treated patient cohorts. This data suggests that mutations in ASXL1 represent novel markers of adverse outcome in myeloid malignancies. We therefore have performed several studies to characterize effects of ASXL1 loss and overexpression of wild-type and mutant ASXL1 on hematopoiesis. To study loss of ASXL1 function, we performed gene expression analyses following shRNA knockdown (KD) of ASXL1 in the ASXL1 wild-type leukemia cell lines, UKE1 and SET2 as well as in CD34+ hematopoietic stem/progenitors. In unbiased analyses we observed significant and reproducible increases in HOXA9, HOXA5, and MLL expression with ASXL1 loss following KD of ASXL1. ChIP (chromatin immunoprecipitation) of HOXA9 and MLL promoters revealed enrichment of the transcriptional activating mark H3K4me3 and decreases in H3K9me3, a repressive mark, at these promoters. ChIP-Seq studies in UKE1 cells with ASXL1 KD similarly reveal a striking decrease in the H3K27me3 repressive mark with ASXL1 loss compared with control cells. Moreover, ChIP using an anti-ASXL1 antibody reveals that ASXL1 itself is present at the promoter of MLL and HOXA5. Our data suggests that ASXL1 mutations (a) promote aberrant hematopoiesis by dysregulated expression of Hox genes and (b) affect target gene expression directly, and by altering the chromatin state of promoters. We propose a series of studies with the following specific aims: Specific Aim #1: Identify target genes of ASXL1 loss and alterations in chromatin state due to ASXL1 loss in normal and leukemic hematopoietic cells using in vitro assays. Specific Aim #2: Determine the in vivo effects of ASXL1 loss on hematopoiesis by targeted disruption of ASXL1. Specific Aim #3: Determine the role of ASXL1 mutations in the pathogenesis of MPNs and AML through studies in patient samples and in in vivo models in cooperation with known MPN and AML disease alleles.